The vascular endothelium releases a variety of vasoactive substances, including the endothelium-derived vasoconstrictor peptide, endothelin (ET) (see, e.g., Vanhoutte et al. (1986) Annual Rev. Physiol. 48: 307-320; Furchgott and Zawadski (1980) Nature 288: 373-376). Endothelin-1, which is a potent twenty-one amino acid peptide vasoconstrictor that was originally identified in the culture supernatant of porcine aortic endothelial cells (see, Yanagisawa et al. (1988) Nature 332: 411-415), is the most potent vasopressor known. It is produced by numerous cell types, including the cells of the endothelium, trachea, kidney and brain. Endothelin is synthesized as a precursor of 203 amino acids, called preproendothelin, containing a signal sequence which is cleaved by an endogenous protease to produce a 38 (human) or 39 (porcine) amino acid peptide. This intermediate, referred to as big endothelin, is processed to the mature biologically active form in vivo by a putative endothelin-converting enzyme (see, e.g., Kashiwabara et al. (1989) FEBS Lttrs. 247: 337-340). Cleavage is required for induction of physiological responses (see, e.g., von Geldern et al. (1991) Peptide Res. 4: 32-35. The endothelin converting enzyme (ECE) appears to be a metal-dependent neutral protease. In porcine aortic endothelial cells, the 39 amino acid intermediate, big endothelin, is hydrolyzed at the Trp.sup.21 --Val.sup.22 bond to generate endothelin-1 and a C-terminal fragment. A similar cleavage occurs in human cells from a 38 amino acid intermediate.
Three distinct endothelin isopeptides, endothelinol, endothelin-2 and endothelin-3, that exhibit potent vasoconstrictor activity have been identified. Each induces vasoconstriction with a potency order of endothelin-2&gt;endothelinol&gt;endothelin-3. In addition, the sarafotoxins (SRTX) S6, a group of peptide toxins from the venom of the snake Atractaspis eingadensis have structural and functional hornology to endothelin-1 and bind competitively to the same cardiac membrane receptors. Sarafotoxins cause severe coronary vasospasm in snake bite victims (Kloog et al. (1989) Trends Pharmacol. Sci. 10: 212-214).
The family of three isopeptides endothelin-1, endothelin-2 and endothelin-3 are encoded by a family of three genes (see, Inoue et al. (1989) Proc. Natl. Acad. Sci. USA 86: 2863-2867; see, also Saida et al. (1989) J. Biol. Chem. 264: 14613-14616). The nucleotide sequences of the three human genes are highly conserved within the region encoding the mature 21 amino acid peptides. Endothelin-2 is (Trp.sup.6,Leu.sup.7) endothelin-1 and endothelino-3 is (Thr.sup.2, Phe.sup.4, Thr.sup.5, Tyr.sup.6, Lys.sup.7, Tyr.sup.14) endothelin-1. These peptides are, thus, highly conserved at the C-terminal ends. In addition, endothelin is highly conserved among species.
Release of endothelins from cultured endothelial cells is modulated by a variety of chemical and physical stimuli and appears to be regulated at the level of transcription and/or translation. For example, gene expression of endothelin-1 is increased by adrenaline, thrombin and Ca.sup.2+ ionophore. The production and release of endothelin from the endothelium is stimulated by angiotensin II, vasopressin and other factors, such as endotoxin and cyclosporin (see, Brooks et al. (1991) Eur. J. Pharm. 194: 115-117), and is inhibited by nitric oxide. Endothelial cells appear to secrete short-lived endothelium-derived relaxing factors (EDRF), such as nitric oxide or a related substance (Palmer et al. (1987) Nature 327: 524-526), when stimulated by vasoactive agents, such as acetylcholine and bradykinin. Endothelin-induced vasoconstriction is also attenuated by atrial natriuretic peptide (ANP).
In vivo and in vitro, the endothelin peptides exhibit numerous biological activities. Endothelin provokes a strong and sustained vasoconstriction in vivo in rats and in isolated vascular smooth muscle preparations; it also provokes the release of eicosanoids and endothelium-derived relaxing factor (EDRF) from perfused vascular beds. Intravenous administration of endothelin-1 and in vitro addition to vascular and other smooth muscle tissues produces long-lasting pressor effects and contraction, respectively (see, e.g., Bolger et al. (1991) Can. J. Physiol. Pharmacol. 69: 406-413). For example, in isolated vascular strips, endothelin-1 is a potent (EC.sub.50 =4.times.10.sup.-10 M) and slow acting, but persistent, contractile agent. In vivo, a single dose elevates blood pressure in about 20 to 30 minutes. In addition to vasoconstriction, endothelin mediates renin release, stimulation of ANP release and induces a positive inotropic action in guinea pig atria. In the lung, endothelin-1 acts as a potent bronchoconstrictor (Maggi et al. (1989) Eur. J. Pharmacol. 160: 179-182). Endothelin increases renal vascular resistance, decreases renal blood flow, and decreases glomerular filtrate rate. It is a potent mitogen of glomerular mesangial cells and invokes the phosphoinoside cascade in such cells (Simonson et al. (1990) J. Clin. Invest. 85: 790-797).
Endothelin-1 plasma levels in healthy individuals, as measured by radioimmunoassay (RIA), are about 0.26-5 pg/ml. Blood levels of endothelin-1 and its precursor, big endothelin, are elevated in shock, myocardial infarction, vasospastic angina, kidney failure and a variety of connective tissue disorders. Increased levels of circulating endothelin are present in patients with pulmonary hypertension. In patients undergoing hemodialysis or kidney transplantation or suffering from cardiogenic shock, myocardial infarction or pulmonary hypertension levels are as high as 35 pg/ml have been observed (see, Stewart et al. (1991) Annals Internal Med. 114: 464-469). The levels of endothelin at the endothelium/smooth muscle interface are probably much higher because endothelin-1 likely acts as a local, rather than a systemic, regulating factor. Because of these numerous physiological effects, endothelin is believed to play a critical role in some pathophysiological conditions, such as hypertension, renal failure, asthma, endotoxin shock and vasospasm (see, Saito et al. (1990) Hypertension 15: 734-738; Tomita et al. (1989) N.EngI.J. Med. 321: 1127; Kurihara et al, (1989) J. Cardiovasc. Pharmacol. 13(Suppl. 5): S13-S17); Morel et al. (1989) Eur. J. Pharmacol. 167: 427-428).
Endothelin-induced vasoconstriction is not affected by antagonists to known neurotransmitters or hormonal factors, but is abolished by calcium channel antagonists. The effect of calcium channel antagonists, however, is most likely the result of blockage of calcium influx, since calcium influx appears to be required for the long-lasting contractile response to endothelin.
There are specific high affinity binding sites (K.sub.d 's in the range of 2-6.times.10.sup.-10 M) for the endothelins in the vascular system and in other tissues, including the intestine, heart, lungs, kidneys, spleen, adrenal glands and brain. Binding is not inhibited by catecholamines, vasoactive peptides, neurotoxins or calcium channel antagonists. Endothelin binds and interacts with receptor sites that are distinct from other autonomic receptors and voltage dependent calcium channels. Competitive binding studies indicate that there are multiple classes of receptors with different affinities for the endothelin isopeptides.
DNA clones encoding two distinct endothelin receptors, designated ET.sub.A and ET.sub.B, have been isolated (Arai et al. (1990) Nature 348: 730-732; Sakurai et al. (1990) Nature 348: 732-735). Based on the amino acid sequence of the proteins encoded by the cloned DNA, it appears that each receptor contains seven membrane spanning domains and exhibits structural similarity to G-protein-coupled membrane proteins. Messenger RNA encoding both receptors has been detected in a variety of tissues, including heart, lung, kidney and brain. The distribution of receptor subtypes is tissue specific (Martin et al. (1989) BioChem. Biophys. Res. Commun. 162: 130-137). ET.sub.A receptors appear to be selective for endothelin-1 and are predominant in cardiovascular tissues. ET.sub.B receptors are predominant in noncardiovascular tissues, including the central nervous system and kidney, and interact with the three endothelin isopeptides (Sakurai et al. (1990) Nature 348: 732-734). In addition, the ET.sub.A receptors, which are endothelin-1-specific, occur on smooth muscle and are linked to vasoconstriction; whereas ET.sub.B receptors are located on the vascular endothelium and are linked to vasodilation (Takayanagi et al. (1991) FEBS Lttrs. 282: 103-106).
The activity of the endothelin isopeptides varies in different tissues by virtue of the distribution of receptor types and the differential affinity of each isopeptide for each receptor type. For example, endothelin-1 inhibits .sup.125 endothelin-1 binding in cardiovascular tissues 40-700 more potently than endothelin-3. .sup.125 Endothelin-1 binding in non-cardiovascular tissues, such as kidney, adrenal gland, and cerebellum, is inhibited to the same extent by endothelin-1 and endothelin-3, which indicates that cardiovascular tissues are rich in ET.sub.A receptors and non-cardiovascular tissues are rich in ET.sub.B receptors.